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Human CLASP1 Mediates Kinetochore Attachment to Dynamic Human CLASP1 mediates kinetochore interactions with the plus ends of dynamic microtubules Helder Maiato*†, Conly L. Rieder‡, Jason R. Swedlow§, Richard Cole‡, Claudio E. Sunkel†║ and William C. Earnshaw*¶ *Chromosome Structure Group, Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, Swann Building, 6.01, University of Edinburgh, King’s Buildings, Mayfield Road, Edinburgh EH9 3JR, Scotland, United Kingdom. †Laboratório de Genética Molecular, Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua Campo Alegre, 823, 4150-180 Porto, Portugal. ‡ Laboratory of Cell Regulation, Division of Molecular Medicine, Wadsworth Center, P.O. Box 509, Albany, New York 12201-0509, USA. §Division of Gene Regulation and Expression, The University of Dundee, MSI/WTB Complex, Dundee, DD1 5EH, Scotland, U. K. ║Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Porto, Portugal. ¶ To whom correspondence should be addressed. Maiato et al. CLASP1 in the kinetochore ABSTRACT One of the most intriguing aspects of mitosis is the ability of kinetochores to hold onto the plus-ends of dynamic microtubules that are actively gaining or losing tubulin subunits. Here we show that the microtubule-associated protein CLASP1 is localized preferentially near the plus-ends of growing microtubules during spindle formation and is also a component of a novel region that we term the outer kinetochore corona. A truncated form of CLASP1 lacking the kinetochore-binding domain behaves as a dominant-negative, leading to the formation of unique single or double asters comprised of radial arrays of microtubule bundles that are highly resistant to depolymerization. Microinjection of cells with antibodies specific to CLASP1 causes bipolar spindles to collapse, forming bipolar or monopolar arrays of microtubules with chromosomes buried in the interior. Suppression of microtubule dynamics in injected cells rescues the kinetochore association with plus ends of microtubules at the periphery of the asters. Our data suggest that CLASP1 is required for kinetochore-associated microtubules to exhibit normal dynamic behaviour. 2 Maiato et al. CLASP1 in the kinetochore INTRODUCTION Kinetochores are the specialized structures that attach chromosomes to the plus ends of spindle microtubules (Brinkley, 1966; Jokelainen, 1967; Euteneuer and McIntosh, 1981). One constraint on this attachment is that microtubules are highly dynamic structures that alternate between states of growth and shrinkage (Mitchison and Kirschner, 1984; Desai and Mitchison, 1997). Furthermore, the bundled kinetochore microtubules exhibit dynamic behaviour whilst remaining attached (Mitchison and Kirschner, 1985; Mitchison et al., 1986; Koshland et al., 1988; Coue et al., 1991; Hyman and Mitchison, 1991). Thus, a captured microtubule is stabilized at a kinetochore not because it becomes less dynamic, but because it cannot detach (Hyman and Karsenti, 1996). The demonstration that kinetochores could hold onto dynamic microtubules identified one of the key questions in chromosome segregation: how do kinetochores remain attached to microtubules that are actively gaining or loosing tubulin subunits at their plus ends? The dynamic behaviour of microtubules is largely regulated by microtubule- associated proteins (MAPs), and recently it has been possible to reconstitute apparently normal microtubule dynamics in solution from purified components (Kinoshita et al., 2001). However, the situation at the kinetochore is more complex, and alternations between states of microtubule growth and shrinkage are regulated at least in part by kinetochore components (Hyman and Mitchison, 1990). Candidates for this role include MCAK/XKCM1 (Wordeman and Mitchison, 1995; Walczak et al., 1996) and the Kin1 kinesins (Desai et al,, 1999), but many other factors are likely to be involved. More recently, extensive studies in budding yeast have pointed to a critical role for a number of non-motor MAPs in kinetochore- microtubule attachment (He et al., 2001; Lin et al., 2001; Cheeseman et al., 2001a; 3 Maiato et al. CLASP1 in the kinetochore Cheeseman et al., 2001b; Janke et al., 2002). This is currently a rapidly advancing area, though difficulties in finding metazoan homologues for several of the gene products have restricted further progress (but see Wigge and Kilmartin, 2001; Howe et al., 2001). The first protein shown to be involved in tethering kinetochores to microtubules was the kinesin-related protein CENP-E (Lombillo et al., 1995), which is now thought to report the status of kinetochore attachment to the metaphase checkpoint (Abrieu et al., 2000). Another protein potentially involved in microtubule plus end binding by the kinetochore is CLIP-170, which was first identified as a factor required for binding of endocytic transport vesicles to microtubules (Pierre et al., 1992). Loss of the S. pombe homolog of CLIP-170, tip1p, results in an increased frequency of microtubule catastrophes (Brunner and Nurse, 2000). The discovery that CLIP-170 localizes at prometaphase (though not metaphase) kinetochores (Dujardin et al., 1998) suggested that the protein might also be important for interactions of kinetochores with microtubules. Subsequent observations appeared to exclude the possibility that CLIP-170 is the prime microtubule-binding factor in the kinetochore (Dujardin et al., 1998), but it remains possible that the protein is important for initial interactions between kinetochores and microtubules. Other microtubule plus-end binding proteins, collectively referred to as +TIPs (Schuyler and Pellman, 2001) have been studied primarily due to their role in promoting polarized cell growth during interphase (McNally, 2001). The CLASP proteins, isolated through their ability to interact with CLIP-170/CLIP-115, were shown to associate with and stabilize microtubule plus ends at the leading edge during fibroblast motility, thereby promoting polarized growth (Akhmanova et al., 2001). Another family of microtubule plus end-binding proteins, the EB1 proteins 4 Maiato et al. CLASP1 in the kinetochore (Su et al., 1995), also appears to be involved in polarized growth of cells apparently through interaction with specific sites on the cell cortex (McNally, 2001). The role of these plus end-binding proteins in mitotic events has been relatively little studied. However APC is found at kinetochores (Kaplan et al., 2001), thereby suggesting a possible role for EB1 in chromosome segregation. A recent RNAi study of Drosophila EB1, confirmed that the protein is required for spindle assembly, particularly stabilization of astral microtubules (Rogers et al., 2002). Kinetochore fibres were detected in EB1-depleted cells, suggesting that this protein does not have an essential role in microtubule binding. Prior to the discovery of the CLASPs, genetic screens had identified a conserved non-motor MAP called variously MAST/Orbit in D. melanogaster (Lemos et al., 2000; Inoue et al., 2000) and Stu1 in S. cerevisiae (Pasqualone and Huffaker, 1994). These proteins are the fly and yeast homologues of the CLASPs. Mitotic phenotypes of MAST/Orbit mutants were complex, but suggested that the protein was essential for spindle assembly and function, possibly by promoting microtubule stability (Kline-Smith and Walczak, 2000). Subsequent RNAi and time-lapse microscopy analysis of MAST alleles revealed that the protein is required for chromosome alignment and maintenance of spindle bipolarity in mitosis (Maiato et al., 2002). The goal of the present work was to determine the role of the human CLASP1 protein in mitotic events. We have shown that CLASP1 is a component of the outer corona of kinetochore that binds to microtubules near their plus ends during mitosis. Interference with CLASP1 function causes the accumulation of monopolar spindles with chromosomes buried in the interior. Our functional analysis of these structures enables us to propose a single simple hypothesis that can explain the multitude of phenotypes seen after perturbation of CLASP1 5 Maiato et al. CLASP1 in the kinetochore function. We propose that CLASP1 is required for the normal regulation of microtubule dynamics at the kinetochore. Thus, CLASP1 is essential for one of the most remarkable and mysterious properties of the kinetochore: the ability to bind and influence the dynamic properties of spindle microtubules. 6 Maiato et al. CLASP1 in the kinetochore RESULTS CLASP1 exhibits dynamic changes in localization during mitosis Endogenous CLASP1 was localized in mitotic HeLa cells using an antibody directed against a peptide sequence specific to CLASP1 (Experimental Procedures). This antibody stained centrosomes and kinetochores in early mitotic cells (Figure 1A, B). Preimmune or peptide competition with the immune sera failed to give any specific staining in mitotic cells (data not shown). CLASP1 relocalized dramatically following the metaphase-anaphase transition, moving to the spindle midzone (Figure 1C), and ultimately concentrating in the midbody (Figure 1D). Staining of the centrosomes remained strong through anaphase, but declined in telophase. This staining pattern was confirmed by expression of GFP-CLASP1 and examination of fixed cells (Figure 1E-H). The pattern of localization by CLASP1 is reminiscent of the localization of CENP-E, a kinesin-related protein that has been implicated in kinetochore attachment to the mitotic spindle and in checkpoint signalling (Yen et al., 1992; Yao et al., 2000; Abrieu et al., 2000). However, the two proteins
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